Pharmaceutical compositions including variants of vascular endothelial cell growth factor having altered pharmacological properties

ABSTRACT

Described herein are pharmaceutical compositions including vascular endothelial cell growth factor (VEGF) variants having modifications in the C-terminal heparin binding domain. The variants exhibit reduced clearance rates for systemic administration generally at lower doses compared with native VEGF thus providing variants having longer availability for therapeutic effect.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. Ser. No. 08/802,052, filedFeb. 14, 1997, now U.S. Pat. No. 6,485,942.

This application contains subject matter related to the following patentapplications: U.S. Ser. No. 08/691,794 filed 2 Aug. 1996, now U.S. Pat.No. 6,057,428; U.S. Ser. No. 08/567,200 filed 5 Dec. 1995, now U.S. Pat.No. 6,020,473; U.S. Ser. No. 60/002,827 filed 25 Aug. 1995; U.S. Ser.No. 07/389,722 filed 4 Aug. 1989 now U.S. Pat. No. 5,332,671; U.S. Ser.No. 07/369,424 filed 21 Jun. 1989, now abandoned; U.S. Ser. No.07/351,117 filed 12 May 1989, now abandoned; U.S. Ser. No. 08/734,443filed 17 Oct. 1996; and U.S. Ser. No. 08/643,839 filed 7 May 1996, nowU.S. Pat. No. 6,100,071.

FIELD OF THE INVENTION

The present invention is directed to particular variants of vascularendothelial cell growth factor (hereinafter sometimes refers to asVEGF), to methods for preparing such variants, and to methods andcompositions and assays utilizing such variants for producingpharmaceutically active materials having therapeutic properties notdiffering at least substantially in kind from the parent compound, VEGF,but having pharmacological properties that differ from the parentcompound, VEGF. In particular, the assays using such variants can beemployed to discover new materials having agonistic or antagonisticproperties to VEGF.

The variants hereof have a demonstrated slower clearance rate comparedwith native material such that they are useful and perhaps saferalternatives for systemic administration as lower doses are availablebecause of such reduced clearance rates; hence, these variants providelonger availability for therapeutic effect.

BACKGROUND OF THE INVENTION

VEGF is a naturally occurring compound that is produced in follicular orfolliculo-stellate cells (FC), a morphologically well characterizedpopulation of granular cells. The FC are stellate cells that sendcytoplasmic processes between secretory cells.

Several years ago a heparin-binding endothelial cell-growth factorcalled vascular endothelial growth factor (VEGF) was identified andpurified from media conditioned by bovine pituitary follicular orfolliculo-stellate cells. See Ferrara et al., Biophys. Res. Comm. 161,851 (1989).

Although a vascular endothelial cell growth factor could be isolated andpurified from natural sources for subsequent therapeutic use, therelatively low concentrations of the protein in FC and the high cost,both in terms of effort and expense, of recovering VEGF provedcommercially unavailing. Accordingly, further efforts were undertaken toclone and express VEGF via recombinant DNA techniques. The embodimentsof that research are set forth in the patent applications referred tosupra; this research was also reported in the scientific literature inLaboratory Investigation 72, 615 (1995), and the references citedtherein.

In those applications there is described an isolated nucleic acidsequence comprising a sequence that encodes a vascular endothelial cellgrowth factor having a molecular weight of about 45,000 daltons undernon-reducing conditions and about 23,000 under reducing conditions asmeasured by SDS-PAGE. Both the DNA and amino acid sequences are setforth in figures forming a part of the present application—see infra.

VEGF prepared as described in the patent applications cited supra, isuseful for treating conditions in which a selected action on thevascular endothelial cells, in the absence of excessive tissue growth,is important, for example, diabetic ulcers and vascular injuriesresulting from trauma such as subcutaneous wounds. Being a vascular(artery and venus) endothelial cell growth factor, VEGF restores cellsthat are damaged, a process referred to as vasculogenesis, andstimulates the formulation of new vessels, a process referred to asangiogenesis.

VEGF is expressed in a variety of tissues as multiple homodimeric forms(121, 165, 189 and 206 amino acids per monomer) resulting fromalternative RNA splicing. VEGF₁₂₁ is a soluble mitogen that does notbind heparin; the longer forms of VEGF bind heparin with progressivelyhigher affinity. The heparin-binding forms of VEGF can be cleaved in thecarboxy terminus by plasmin to release (a) diffusible form(s) of VEGF.Amino acid sequencing of the carboxy terminal peptide identified afterplasmin cleavage is Arg₁₁₀–Ala₁₁₁. Amino terminal “core” protein, VEGF(1-110) isolated as a homodimer, binds neutralizing monoclonalantibodies (4.6.1 and 2E3) and soluble forms of FLT-1, KDR and FLKreceptors with similar affinity compared to the intact VEGF₁₆₅homodimer. VEGF contains a C-terminal heparin binding domain thatgenerally spans the C-terminus beginning beyond about amino acid 120.Generally this domain carries a relatively large number of positivelycharged amino acids.

The present invention is predicated upon initial research results thatcompared the heparin binding properties of VEGF derived respectivelyfrom recombinant Chinese hamster ovary (CHO) and E. coli cells. Thisresearch resulted in the finding that the CHO-derived VEGF materialcontained various C-terminal “processing” resulting in forms havingdifferent lengths with respect to the heparin binding C-terminal domainversus the substantially full-length material derived via E. coliproduction. Such C-terminal processing includes internal clips, i.e.,cleaved sites, within the heparin binding domain that may alter thesecondary and tertiary structure of the heparin binding domain so as todecease its affinity for heparin and endogenous heparan sulfateproteoglycans.

Further research indicated that such C-terminal processing of VEGFresulted in variants exhibiting slower rates of clearance and smallervolumes of distribution. Although these processed variants still possessat least a portion of the heparin binding domain, the modified domainbound heparin and heparin sulfate proteoglycans with lower affinity. Theresultant effect was that less VEGF was cleared by non-specific targetorgans such as the liver.

It was therefore a further object of this research to produce VEGFvariants that would have C-terminal variations with consequentialvarying heparin binding affinity resulting in variants of VEGF having areduced clearance rate and hence longer retention within the body aftersystemic administration such that lower doses of the material wereavailable for systemic administration for therapeutic effect. Theintellectual property of such research is the subject of the presentinvention.

SUMMARY OF THE INVENTION

The objects of this invention, as defined generally supra, are achievedby the provision of vascular endothelial cell growth factor (VEGF)variants having modifications in the C-terminus heparin binding domain,said variants exhibiting reduced clearance rates for systemicadministration generally at lower doses compared with native VEGF thusproviding variants having longer availability for therapeutic effect.

In a preferred embodiment, such modifications result in structuralalterations effected within the region of the C-terminus heparin bindingdomain bridging about amino acid 121 to about amino acid 165, and morepreferably around the protease sensitive sites at positions 125 and 147and/or at other sites within the domain where structural alterationsalter functional binding characteristics, such as at the loci ofpositively charged amino acids.

The variants hereof may be prepared via recombinant DNA technologytaking advantage of the available tools and techniques for providing DNAhaving deletions in the C-terminal domain such that the recombinantexpression of such DNA provides VEGF variants wherein the C-terminusheparin binding domain contain deletions with resultant alteredpharmacological properties affecting therapeutic results. Alternatively,such variants can be isolated from recombinant systems that causeproteolytic cleavage at the C-terminus (a largely basic amino acidcontaining domain) resulting in C-terminus heparin binding domaindeletion variants. Alternatively, such C-terminus deletion variants areproducts of for example carboxypeptidase B treatment.

In other aspects, the present invention relates to DNA sequencesencoding the various variants described supra, replicable expressionvectors capable of expressing said DNA sequences via transforming DNA ina transformant host cell, and microorganisms and cell cultures which aretransformed with such vectors.

In further aspects hereof, the present invention is directed to methodsuseful for the recombinant expression of such DNA referred to aboveincluding methods of isolation and purification as a part of recovery.

In yet further aspects, the present invention is directed tocompositions useful for treating indications where vasculogenesis orangiogenesis is desired for treatment of an underlying disease statecomprising a therapeutically effective amount of a VEGF variant hereof,advantageously being reduced in general dosage form because of thereduced clearance rates exhibited by the variants hereof, in admixturewith a pharmaceutically acceptable carrier. Thus, the present inventionprovides variants of VEGF wherein their exhibited reduced rate ofclearance provides resultant effects that provide in turn saferalternative systemic administration with lower doses resulting in longeravailability for therapeutic effect. In addition, a decrease inpotential toxic side effects would be expected due to the decrease innonspecific binding of the heparin domain modified variants tonon-target tissues.

Thus, the present invention is directed to VEGF C-terminal heparinbinding domain variants having structural alterations that result infunctional modification of the heparin binding characteristics of thatVEGF variant molecule. Such structural alterations can be imparted by,for example, internal cleavage at various proteolytic sites and/or byvarious mutations. For example structural alterations may change theionic charge of the domain by replacement of the largely positivelycharged amino acids with negatively or neutrally charged amino acidsand/or by other mutations resulting in derivatives such as by deletionsof amino acids, substitutions, and so forth.

All such structural alterations are believed to result in molecules thathave in turn altered conformational structure that affects the heparinbinding characteristics. It is believed that because the heparin bindingdomain is a highly positively charged domain, the bindingcharacteristics with heparin are probably an ionic interaction.Therefore, structural alterations that would affect ionic interactionwould in turn affect binding. Thus, any such structural alterations thatresult in affecting heparin binding are covered within the scope of thepresent invention, i.e., such VEGF variants hereof manifest functionaleffect.

Expanding on the basic premise hereof based upon the finding of theeffects of C-terminus heparin binding domain deletion variants of VEGF,the present invention is directed to all associated embodiments derivingtherefrom, including recombinant DNA materials and processes forpreparing such variants, materials and information for compounding suchvariants into pharmaceutically finished form and various assays usingsuch variants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict both the amino acid (SEQ ID NO:2) and DNAsequence (SEQ ID NO:1) for VEGF having 165 amino acids. Predicted aminoacids of the protein are shown below the DNA sequence and are numberedfrom the first residue of the N-terminus of the protein sequence.Negative amino acid numbers refer to the presumed leader signal sequenceor pre-protein, while positive numbers refer to the putative matureprotein.

FIG. 2 depicts schematics of VEGF which include the C-terminus heparinbinding domain of VEGF. The heparin binding domain is generallyconsidered to be located from about amino acid 121 to the C-terminalamino acid of the full-length molecule (SEQ ID NO:3). The top panel ofFIG. 2 shows a plasmin cleavage site and the bottom panel of FIG. 2shows potential disulfide bonds between cysteine residues and clip sitesat residues 110, 125 and 147.

FIG. 3 depicts the construction with its various elements of the plasmidpSDVF₁₆₅.

FIG. 4 depicts the construction with its various elements of the plasmidpRK5.

FIG. 5 depicts the construction schematic followed to prepare the finalexpression vectors harboring DNA that contains mutations in accordancewith preparing the various products of the present invention.

FIG. 6 depicts the comparative pharmacokinetic (PK) study between E.coli and CHO VEGF in rats. It can be seen that the VEGF obtained from E.coli cleared more rapidly due to increased binding to heparin orendogenous heparin sulfate proteoglycans.

FIG. 7 depicts the calculated pharmacokinetic parameters for CHO versusE. Coli VEGF as given in FIG. 6 and FIG. 8, respectively.

FIG. 8 depicts [125I]—VEGF in isolated rat liver perfusion.

FIG. 9 depicts E. coli and CHO VEGF comparison by heparin columnchromotography.

FIG. 10 depicts heparin chromatography of various VEGF samples.

FIG. 11 depicts the results of E. coli-derived VEGF aftercarboxypeptodase treatment compared with CHO-derived VEGF.

FIG. 12 depicts the CHO-derived VEGF peaks 1 and 2 by liver perfusion.

FIG. 13 displays the in vivo pharmacokinetics of E. coli-derived VEGF(control, peaks 1 and 2).

FIG. 14 displays the in vivo pharmacokinetics of E. coli-derived VEGF(control, peaks 1 and 2).

FIG. 15 depicts the SDS Page gel results of various iodinated VEGFsamples.

FIG. 16 shows the effect of VEGF variants on ACE cell proliferation.

FIGS. 17A–C depict data showing VEGF binding to red blood cells ofvarious mammal species. FIG. 17A depicts results obtained from rat bloodcells, FIG. 17B depicts results obtained from rabbit blood cells, andFIG. 17C depicts results obtained from human blood cells.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

As used herein, “vascular endothelial cell growth factor,” or “VEGF,”refers to a mammalian growth factor as defined in U.S. Pat. No.5,332,671, including the human amino acid sequence of FIGS. 1A and 1B.The biological activity of native VEGF is shared by any analogue orvariant thereof that is capable of promoting selective growth ofvascular endothelial cells but not of bovine corneal endothelial cells,lens epithelial cells, adrenal cortex cells, BHK-21 fibroblasts, orkeratinocytes, or that possesses an immune epitope that isimmunologically cross-reactive with an antibody raised against at leastone epitope of the corresponding native VEGF.

The term “variant” refers to VEGF molecules that contain amodification(s) in the C-terminus heparin binding domain that results infunctional modification of the pharmacokinetic profile, that is, theproduction of a molecule having a reduced clearance rate compared withnative material. It is believed that such modifications affect theconfirmational structure of the resultant variant, hence the use of theterm “structural alteration” in respect of such “modifications.”. Thesemodifications may be the result of DNA mutagenesis so as to createmolecules having different amino acids from those found in the nativematerial. In particular, as the C-terminus heparin binding domaincontains a relatively large number of positively charged amino acids,the binding of that domain with heparin is believed to be based uponionic interactions. Accordingly, preferred embodiments would replacepositively charged amino acids with negatively or neutrally chargedamino acids. Alternatively, the molecule may be modified by deletingportions of the C-terminal heparin binding domain. Still alternatively,proteolytic cleavage of the C-terminal heparin binding domain at variouscleavage sites result in VEGF variants that have modifications resultingin reduced clearance rates. Thus, the present invention is directed toany modification to the C-terminal heparin binding domain of VEGF thatresults in a molecule that has affected pharmacokinetic properties i.e.,a functional definition.

“Transfection” refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

“Transformation” means introducing DNA into an organism so that the DNAis replicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described by Cohen, S.N. Proc. Natl.Acad. Sci. (USA), 69, 2110 (1972) and Mandel et al. J. Mol. Biol. 53,154 (1970), is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. For mammalian cells without suchcell walls, the calcium phosphate precipitation method of Graham, F. andvan der Eb, A., Virology, 52, 456–457 (1978) is preferred. Generalaspects of mammalian cell host system transformations have beendescribed by Axel in U.S. Pat. No. 4,399,216 issued Aug. 16, 1983.Transformations into yeast are typically carried out according to themethod of Van Solingen, P., et al. J. Bact., 130, 946 (1977) and Hsiao,C. L., et al. Proc. Natl. Acad. Sci. (USA) 76, 3829 (1979). However,other methods for introducing DNA into cells such as by nuclearinjection or by protoplast fusion may also be used.

“Site-directed mutagenesis” is a technique standard in the art, and isconducted using a synthetic oligonucleotide primer complementary to asingle-stranded phage DNA to be mutagenized except for limitedmismatching, representing the desired mutation. Briefly, the syntheticoligonucleotide is used as a primer to direct synthesis of a strandcomplementary to the phage, and the resulting double-stranded DNA istransformed into a phage-supporting host bacterium. Cultures of thetransformed bacteria are plated in top agar, permitting plaque formationfrom single cells that harbor the phage. Theoretically, 50% of the newplaques will contain the phage having, as a single strand, the mutatedform; 50% will have the original sequence. The plaques are hybridizedwith kinased synthetic primer at a temperature that permitshybridization of an exact match, but at which the mismatches with theoriginal strand are sufficient to prevent hybridization. Plaques thathybridize with the probe are then selected and cultured, and the DNA isrecovered.

“Operably linked” refers to juxtaposition such that the normal functionof the components can be performed. Thus, a coding sequence “operablylinked” to control sequences refers to a configuration wherein thecoding sequence can be expressed under the control of these sequencesand wherein the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, then synthetic oligonucleotide adaptors orlinkers are used in accord with conventional practice.

“Control sequences” refers to DNA sequences necessary for the expressionof an operably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, a ribosome bindingsite, and possibly, other as yet poorly understood sequences. Eukaryoticcells are known to utilize promoters, polyadenylation signals, andenhancers.

“Expression system” refers to DNA sequences containing a desired codingsequence and control sequences in operable linkage, so that hoststransformed with these sequences are capable of producing the encodedproteins. To effect transformation, the expression system may beincluded on a vector; however, the relevant DNA may then also beintegrated into the host chromosome.

As used herein, “cell,” “cell line,” and “cell culture” are usedinterchangeably and all such designations include progeny. Thus,“transformants” or “transformed cells” includes the primary subject celland cultures derived therefrom without regard for the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.Mutant progeny that have the same functionality as screened for in theoriginally transformed cell are included. Where distinct designationsare intended, it will be clear from the context.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein arecommercially available, are publicly available on an unrestricted basis,or can be constructed from such available plasmids in accord withpublished procedures. In addition, other equivalent plasmids are knownin the art and will be apparent to the ordinary artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with anenzyme that acts only at certain locations in the DNA. Such enzymes arecalled restriction enzymes, and the sites for which each is specific iscalled a restriction site. The various restriction enzymes used hereinare commercially available and their reaction conditions, cofactors, andother requirements as established by the enzyme suppliers are used.Restriction enzymes commonly are designated by abbreviations composed ofa capital letter followed by other letters representing themicroorganism from which each restriction enzyme originally was obtainedand then a number designating the particular enzyme. In general, about 1mg of plasmid or DNA fragment is used with about 1–2 units of enzyme inabout 20 ml of buffer solution. Appropriate buffers and substrateamounts for particular restriction enzymes are specified by themanufacturer. Incubation of about 1 hour at 37° C. is ordinarily used,but may vary in accordance with the supplier's instructions. Afterincubation, protein is removed by extraction with phenol and chloroform,and the digested nucleic acid is recovered from the aqueous fraction byprecipitation with ethanol. Digestion with a restriction enzymeinfrequently is followed with bacterial alkaline phosphatase hydrolysisof the terminal 5′ phosphates to prevent the two restriction cleavedends of a DNA fragment from “circularizing” or forming a closed loopthat would impede insertion of another DNA fragment at the restrictionsite. Unless otherwise stated, digestion of plasmids is not followed by5′ terminal dephosphorylation. Procedures and reagents fordephosphorylation are conventional (T. Maniatis et al. 1982, MolecularCloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory,1982) pp.133–134).

“Recovery” or “isolation” of a given fragment of DNA from a restrictiondigest means separation of the digest on polyacrylamide or agarose gelby electrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. This procedure is knowngenerally. For example, see R. Lawn et al., Nucleic Acids Res. 9,6103–6114 (1981), and D. Goeddel et al., Nucleic Acids Res. 8, 4057(1980).

“Southern Analysis” is a method by which the presence of DNA sequencesin a digest or DNA-containing composition is confirmed by hybridizationto a known, labeled oligonucleotide or DNA fragment. For the purposesherein, unless otherwise provided, Southern analysis shall meanseparation of digests on 1 percent agarose, denaturation, and transferto nitrocellulose by the method of E. Southern, J. Mol. Biol. 98,503–517 (1975), and hybridization as described by T. Maniatis et al.,Cell 15, 687–701 (1978).

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (T. Maniatis et al. 1982,supra, p.146). Unless otherwise provided, ligation may be accomplishedusing known buffers and conditions with 10 units of T4 DNA ligase(“ligase”) per 0.5 mg of approximately equimolar amounts of the DNAfragments to be ligated.

“Preparation” of DNA from transformants means isolating plasmid DNA frommicrobial culture. Unless otherwise provided, the alkaline/SDS method ofManiatis et al. 1982, supra, p. 90, may be used.

“Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as described in EP Pat. Pub. No. 266,032published May 4, 1988, or via deoxynucleoside H-phosphonateintermediates as described by Froehler et al., Nucl. Acids Res. 14,5399–5407[1986]). They are then purified on polyacrylamide gels.

B. General Methodology

1. Glycosylation

The VEGF amino acid sequence variant may contain at least one amino acidsequence that has the potential to be glycosylated through an N-linkageand that is not normally glycosylated in the native molecule.

2. Amino Acid Sequence Variants

a. Additional Mutations

For purposes of shorthand designation of VEGF variants described herein,it is noted that numbers refer to the amino acid residue/position alongthe amino acid sequences of putative mature VEGF. Amino acididentification uses the single-letter alphabet of amino acids, i.e.,

Asp D Aspartic acid Ile I Isoleucine Thr T Threonine Leu L Leucine Ser SSerine Tyr Y Tyrosine Glu E Glutamic acid Phe F Phenylalanine Pro PProline His H Histidine Gly G Glycine Lys K Lysine Ala A Alanine Arg RArginine Cys C Cysteine Trp W Tryptophan Val V Valine Gln Q GlutamineMet M Methionine Asn N Asparagine

The present invention is directed to C-terminus heparin binding domainvariants of VEGF. These variants forming the predicate of the presentinvention may also contain additional variations within the backbone ofthe VEGF molecule which does not affect the biological properties of theVEGF fundamental variant hereof in kind.

It will be appreciated that those certain other variants at otherpositions in the VEGF molecule can be made without departing from thespirit of the present invention with respect to the C-terminus heparinbinding domain deletion variations hereof. Thus, point mutational orother broader variations may be made in all other parts of the moleculeso as to impart interesting properties that again do not affect theoverall properties of the fundamental variant with respect to theC-terminal deletion. These latter, additional variants may be made bymeans generally known in the art. For example covalent modifications maybe made to various of the amino acid residues.

Cysteinyl residues most commonly are reacted with a-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,a-bromo-b-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5–7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing a-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneare used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively. Tyrosyl residues are iodinated using ₁₂₅I or ₁₃₁I toprepare labeled proteins for use in radioimmunoassay, the chloramine Tmethod described above being suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′-N-C-N-R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking theVEGF to a water-insoluble support matrix or surface for use in themethod for purifying anti-VEGF antibodies. Commonly used crosslinkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxy-succinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the a-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79–86[1983]),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl group.

b. DNA Mutations

Amino acid sequence variants of VEGF can also be prepared by mutationsin the DNA. Such variants include, for example, deletions from, orinsertions or substitutions of, residues within the amino acid sequenceshown in FIGS. 1A and 1B. Any combination of deletion, insertion, andsubstitution may also be made to arrive at the final construct, providedthat the final construct possesses the desired activity. Obviously, themutations that will be made in the DNA encoding the variant must notplace the sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure (seeEP 75,444A).

At the genetic level, these variants ordinarily are prepared bysite-directed mutagenesis of nucleotides in the DNA encoding the VEGF,thereby producing DNA encoding the variant, and thereafter expressingthe DNA in recombinant cell culture. The variants typically exhibit thesame qualitative biological activity as the naturally occurring analog.

While the site for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, to optimize the performance of a mutation at a given site,random mutagenesis may be conducted at the target codon or region andthe expressed VEGF variants screened for the optimal combination ofdesired activity. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, forexample, site-specific mutagenesis.

Preparation of VEGF variants in accordance herewith is preferablyachieved by site-specific mutagenesis of DNA that encodes an earlierprepared variant or a nonvariant version of the protein. Site-specificmutagenesis allows the production of VEGF variants through the use ofspecific oligonucleotide sequences that encode the DNA sequence of thedesired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 20 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered. In general, thetechnique of site-specific mutagenesis is well known in the art, asexemplified by publications such as Adelman et al., DNA 2, 183 (1983),the disclosure of which is incorporated herein by reference.

As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), thedisclosure of which is incorporated herein by reference. These phage arereadily commercially available and their use is generally well known tothose skilled in the art. Alternatively, plasmid vectors that contain asingle-stranded phage origin of replication (Veira et al., Meth.Enzymol., 153, 3[1987]) may be employed to obtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector that includeswithin its sequence a DNA sequence that encodes the relevant protein. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically, for example, by the method of Crea et al.,Proc. Natl. Acad. Sci. (USA), 75, 5765 (1978). This primer is thenannealed with the single-stranded protein-sequence-containing vector,and subjected to DNA-polymerizing enzymes such as E. coli polymerase IKlenow fragment, to complete the synthesis of the mutation-bearingstrand. Thus, a heteroduplex is formed wherein one strand encodes theoriginal non-mutated sequence and the second strand bears the desiredmutation. This heteroduplex vector is then used to transform appropriatecells such as JM101 cells and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.

After such a clone is selected, the mutated protein region may beremoved and placed in an appropriate vector for protein production,generally an expression vector of the type that may be employed fortransformation of an appropriate host.

c. Types of Mutations

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably 1 to 10 residues, and typically arecontiguous.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions of from one residue to polypeptides of essentially unrestrictedlength, as well as intrasequence insertions of single or multiple aminoacid residues. Intrasequence insertions (i.e., insertions within themature VEGF sequence) may range generally from about 1 to 10 residues,more preferably 1 to 5. An example of a terminal insertion includes afusion of a signal sequence, whether heterologous or homologous to thehost cell, to the N-terminus of the VEGF molecule to facilitate thesecretion of mature VEGF from recombinant hosts.

The third group of variants are those in which at least one amino acidresidue in the VEGF molecule, and preferably only one, has been removedand a different residue inserted in its place. Such substitutionspreferably are made in accordance with the following Table 1 when it isdesired to modulate finely the characteristics of a VEGF molecule.

TABLE 1 Original Residue Exemplary Substitutions Ala (A) gly; ser Arg(R) lys Asn (N) gln; his Asp (D) glu Cys (C) ser Gln (Q) asn Glu (E) aspGly (G) ala; pro His (H) asn; gln Ile (I) leu; val Leu (L) ile; val Lys(K) arg; gln; glu Met (M) leu; tyr; ile Phe (F) met; leu; tyr Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in TableI, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. The substitutions thatin general are expected to produce the greatest changes in VEGFproperties will be those in which (a) glycine and/or proline (P) issubstituted by another amino acid or is deleted or inserted; (b) ahydrophilic residue, e.g., seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, oralanyl; (c) a cysteine residue is substituted for (or by) any otherresidue; (d) a residue having an electropositive side chain, e.g.,lysyl, arginyl, or histidyl, is substituted for (or by) a residue havingan electronegative charge, e.g., glutamyl or aspartyl; (e) a residuehaving an electronegative side chain is substituted for (or by) aresidue having an electropositive charge; or (f) a residue having abulky side chain, e.g., phenylalanine, is substituted for (or by) onenot having such a side chain, e.g., glycine.

Most deletions and insertions, and substitutions in particular, are notexpected to produce radical changes in the characteristics of the VEGFmolecule. However, when it is difficult to predict the exact effect ofthe substitution, deletion, or insertion in advance of doing so, oneskilled in the art will appreciate that the effect will be evaluated byroutine screening assays. For example, a variant typically is made bysite-specific mutagenesis of the native VEGF-encoding nucleic acid,expression of the variant nucleic acid in recombinant cell culture, and,optionally, purification from the cell culture, for example, byimmunoaffinity adsorption on a rabbit polyclonal anti-VEGF column (toabsorb the variant by binding it to at least one remaining immuneepitope).

Since VEGF tends to aggregate into dimers, it is within the scope hereofto provide hetero- and homodimers, wherein one or both subunits arevariants. Where both subunits are variants, the changes in amino acidsequence can be the same or different for each subunit chain.Heterodimers are readily produced by cotransforming host cells with DNAencoding both subunits and, if necessary, purifying the desiredheterodimer, or by separately synthesizing the subunits, dissociatingthe subunits (e.g., by treatment with a chaotropic agent such as urea,guanidine hydrochloride, or the like), mixing the dissociated subunits,and then reassociating the subunits by dialyzing away the chaotropicagent.

Also included within the scope of mutants herein are so-calledglyco-scan mutants. This embodiment takes advantage of the knowledge ofso-called glycosylation sites. Thus, where appropriate such aglycosylation site can be introduced so as to produce a speciescontaining glycosylation moieties at that position. Similarly, anexisting glycosylation site can be removed by mutation so as to producea species that is devoid of glycosylation at that site. It will beunderstood, again, as with the other mutations contemplated by thepresent invention, that they are introduced within the so-called KDRand/or FLT-1 domains in accord with the basic premise of the presentinvention, and they can be introduced at other locations outside ofthese domains within the overall molecule so long as the final productdoes not differ in overall kind from the properties of the mutationintroduced in one or both of said two binding domains.

The activity of the cell lysate or purified VEGF variant is thenscreened in a suitable screening assay for the desired characteristic.For example, a change in the immunological character of the VEGFmolecule, such as affinity for a given antibody, is measured by acompetitive-type immunoassay. Changes in the enhancement or suppressionof vascular endothelium growth by the candidate mutants are measured bythe appropriate assay. Modifications of such protein properties as redoxor thermal stability, hydrophobicity, susceptibility to proteolyticdegradation, or the tendency to aggregate with carriers or intomultimers are assayed by methods well known to the ordinarily skilledartisan.

3. Recombinant Expression

The VEGF molecule desired may be prepared by any technique, includingrecombinant methods. Likewise, an isolated DNA is understood herein tomean chemically synthesized DNA, cDNA, chromosomal, or extrachromosomalDNA with or without the 3′- and/or 5′-flanking regions. Preferably, thedesired VEGF herein is made by synthesis in recombinant cell culture.

For such synthesis, it is first necessary to secure nucleic acid thatencodes a VEGF. DNA encoding a VEGF molecule may be obtained frombovine, pituitary follicular cells by (a) preparing a cDNA library fromthese cells, (b) conducting hybridization analysis with labeled DNAencoding the VEGF or fragments thereof (up to or more than 100 basepairs in length) to detect clones in the library containing homologoussequences, and (c) analyzing the clones by restriction enzyme analysisand nucleic acid sequencing to identify full-length clones. DNA that iscapable of hybridizing to a VEGF-encoding DNA under low stringencyconditions is useful for identifying DNA encoding VEGF. Both high andlow stringency conditions are defined further below. If full-lengthclones are not present in a cDNA library, then appropriate fragments maybe recovered from the various clones using the nucleic acid sequenceinformation disclosed herein for the first time and ligated atrestriction sites common to the clones to assemble a full-length cloneencoding the VEGF. Alternatively, genomic libraries will provide thedesired DNA. The sequence of the DNA encoding human VEGF that wasultimately determined by probing a human leukemia cell line is shown inFIGS. 1A and 1B.

Once this DNA has been identified and isolated from the library it isligated into a replicable vector for further cloning or for expression.

In one example of a recombinant expression system a VEGF-encoding geneis expressed in mammalian cells by transformation with an expressionvector comprising DNA encoding the VEGF. It is preferable to transformhost cells capable of accomplishing such processing so as to obtain theVEGF in the culture medium or periplasm of the host cell, i.e., obtain asecreted molecule.

a. Useful Host Cells and Vectors

The vectors and methods disclosed herein are suitable for use in hostcells over a wide range of prokaryotic and eukaryotic organisms.

In general, of course, prokaryotes are preferred for the initial cloningof DNA sequences and construction of the vectors useful in theinvention. For example, E coli K12 strain MM 294 (ATCC No. 31,446) isparticularly useful. Other microbial strains that may be used include E.coli strains such as E. coli B and E. coli X 1776 (ATCC No. 31,537).These examples are, of course, intended to be illustrative rather thanlimiting.

Prokaryotes may also be used for expression. The aforementioned strains,as well as E. coli strains W3110 (F-, lambda-, prototrophic, ATCC No.27,325), K5772 (ATCC No. 53,635), and SR101, bacilli such as Bacillussubtilis, and other enterobacteriaceae such as Salmonella typhimurium orSerratia marcesans, and various pseudomonas species, may be used.

In general, plasmid vectors containing replicon and control sequencesthat are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences that are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies (see, e.g., Bolivar et al., Gene 2, 95[1977]). pBR322 containsgenes for ampicillin and tetracycline resistance and thus provides easymeans for identifying transformed cells. The pBR322 plasmid, or othermicrobial plasmid or phage, must also contain, or be modified tocontain, promoters that can be used by the microbial organism forexpression of its own proteins.

Those promoters most commonly used in recombinant DNA constructioninclude the b-lactamase (penicillinase) and lactose promoter systems(Chang et al., Nature, 375, 615[1978]; Itakura et al., Science, 198,1056[1977]; Goeddel et al., Nature, 281, 544[1979]) and a tryptophan(trp) promoter system (Goeddel et al., Nucleic Acids Res., 8,4057[1980]; EPO Appl. Publ. No. 0036,776). While these are the mostcommonly used, other microbial promoters have been discovered andutilized, and details concerning their nucleotide sequences have beenpublished, enabling a skilled worker to ligate them functionally withplasmid vectors (see, e.g., Siebenlist et al., Cell, 20, 269[1980]).

In addition to prokaryotes, eukaryotic microbes, such as yeast cultures,may also be used. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among eukaryotic microorganisms, although anumber of other strains are commonly available. For expression inSaccharomyces, the plasmid YRp7, for example (Stinchcomb et al., Nature282, 39[1979]; Kingsman et al., Gene 7,141[1979]; Tschemper et al., Gene10, 157[1980]), is commonly used. This plasmid already contains the trp1gene that provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44,076or PEP4-1 (Jones, Genetics, 85, 12[1977]). The presence of the trp1lesion as a characteristic of the yeast host cell genome then providesan effective environment for detecting transformation by growth in theabsence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255,2073[1980]) or other glycolytic enzymes (Hess et al., J. Adv. EnzymeReg. 7,149[1968]; Holland et al., Biochemistry 17, 4900[1978]), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructo-kinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination. Other promoters, which have the additional advantage oftranscription controlled by growth conditions, are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Any plasmid vectorcontaining yeast-compatible promoter, origin of replication andtermination sequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years [Tissue Culture, Academic Press, Kruseand Patterson, editors (1973)]. Examples of such useful host cell linesare VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, andW138, BHK, COS-7, 293, and MDCK cell lines. Expression vectors for suchcells ordinarily include (if necessary) an origin of replication, apromoter located in front of the gene to be expressed, along with anynecessary ribosome binding sites, RNA splice sites, polyadenylationsites, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus2, and most frequentlySimian Virus 40 (SV40). The early and late promoters of SV40 virus areparticularly useful because both are obtained easily from the virus as afragment that also contains the SV40 viral origin of replication [Fierset al., Nature, 273, 113 (1978)]. Smaller or larger SV40 fragments mayalso be used, provided there is included the approximately 250-bpsequence extending from the HindIII site toward the BgII site located inthe viral origin of replication. Further, it is also possible, and oftendesirable, to utilize promoter or control sequences normally associatedwith the desired gene sequence, provided such control sequences arecompatible with the host cell systems.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

Satisfactory amounts of protein are produced by cell cultures; however,refinements, using a secondary coding sequence, serve to enhanceproduction levels even further. One secondary coding sequence comprisesdihydrofolate reductase (DHFR) that is affected by an externallycontrolled parameter, such as methotrexate (MTX), thus permittingcontrol of expression by control of the methotrexate concentration.

In selecting a preferred host cell for transfection by the vectors ofthe invention that comprise DNA sequences encoding both VEGF and DHFRprotein, it is appropriate to select the host according to the type ofDHFR protein employed. If wild-type DHFR protein is employed, it ispreferable to select a host cell that is deficient in DHFR, thuspermitting the use of the DHFR coding sequence as a marker forsuccessful transfection in selective medium that lacks hypoxanthine,glycine, and thymidine. An appropriate host cell in this case is theChinese hamster ovary (CHO) cell line deficient in DHFR activity,prepared and propagated as described by Urlaub and Chasin, Proc. Natl.Acad. Sci. (USA) 77, 4216 (1980).

On the other hand, if DHFR protein with low binding affinity for MTX isused as the controlling sequence, it is not necessary to useDHFR-deficient cells. Because the mutant DHFR is resistant tomethotrexate, MTX-containing media can be used as a means of selectionprovided that the host cells are themselves methotrexate sensitive. Mosteukaryotic cells that are capable of absorbing MTX appear to bemethotrexate sensitive. One such useful cell line is a CHO line, CHO-K1(ATCC No. CCL 61).

b. Typical Methodology Employable

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to prepare the plasmids required.

If blunt ends are required, the preparation may be treated for 15minutes at 15° C. with 10 units of Polymerase I (Klenow),phenol-chloroform extracted, and ethanol precipitated.

Size separation of the cleaved fragments may be performed using 6percent polyacrylamide gel described by Goeddel et al., Nucleic AcidsRes. 8, 4057 (1980).

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are typically used to transform E. coli K12 strain 294(ATCC 31,446) or other suitable E. coli strains, and successfultransformants selected by ampicillin or tetracycline resistance whereappropriate. Plasmids from the transformants are prepared and analyzedby restriction mapping and/or DNA sequencing by the method of Messing etal., Nucleic Acids Res. 9, 309 (1981) or by the method of Maxam et al.,Methods of Enzymology 65, 499 (1980).

After introduction of the DNA into the mammalian cell host and selectionin medium for stable transfectants, amplification of DHFR-protein-codingsequences is effected by growing host cell cultures in the presence ofapproximately 20,000–500,000 nM concentrations of methotrexate, acompetitive inhibitor of DHFR activity. The effective range ofconcentration is highly dependent, of course, upon the nature of theDHFR gene and the characteristics of the host. Clearly, generallydefined upper and lower limits cannot be ascertained. Suitableconcentrations of other folic acid analogs or other compounds thatinhibit DHFR could also be used. MTX itself is, however, convenient,readily available, and effective.

Other techniques employable are described in a section just prior to theexamples.

4. Utilities and Formulation

The VEGF molecules herein have a number of therapeutic uses associatedwith the vascular endothelium. Such uses include the treatment oftraumata to the vascular network, in view of the demonstrated rapidpromotion by VEGF of the proliferation of vascular endothelial cellsthat would surround the traumata. Examples of such traumata that couldbe so treated include, but are not limited to, surgical incisions,particularly those involving the heart, wounds, including lacerations,incisions, and penetrations of blood vessels, and surface ulcersinvolving the vascular endothelium such as diabetic, hemophiliac, andvaricose ulcers. Other physiological conditions that could be improvedbased on the selective mitogenic character of VEGF are also includedherein.

For the traumatic indications referred to above, the VEGF molecule willbe formulated and dosed in a fashion consistent with good medicalpractice taking into account the specific disorder to be treated, thecondition of the individual patient, the site of delivery of the VEGF,the method of administration, and other factors known to practitioners.Thus, for purposes herein, the “therapeutically effective amount” of theVEGF is an amount that is effective either to prevent, lessen theworsening of, alleviate, or cure the treated condition, in particularthat amount which is sufficient to enhance the growth of vascularendothelium in vivo.

VEGF amino acid sequence variants and derivatives that areimmunologically crossreactive with antibodies raised against native VEGFare useful in immunoassays for VEGF as standards, or, when labeled, ascompetitive reagents.

The VEGF is prepared for storage or administration by mixing VEGF havingthe desired degree of purity with physiologically acceptable carriers,excipients, or stabilizers. Such materials are non-toxic to recipientsat the dosages and concentrations employed. If the VEGF is watersoluble, it may be formulated in a buffer such as phosphate or otherorganic acid salt preferably at a pH of about 7 to 8. If a VEGF variantis only partially soluble in water, it may be prepared as amicroemulsion by formulating it with a nonionic surfactant such asTween, Pluronics, or PEG, e.g., Tween 80, in an amount of 0.04–0.05%(w/v), to increase its solubility.

Optionally other ingredients may be added such as antioxidants, e.g.,ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; and sugar alcohols such asmannitol or sorbitol.

The VEGF to be used for therapeutic administration must be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). The VEGF ordinarilywill be stored in lyophilized form or as an aqueous solution if it ishighly stable to thermal and oxidative denaturation. The pH of the VEGFpreparations typically will be about from 6 to 8, although higher orlower pH values may also be appropriate in certain instances. It will beunderstood that use of certain of the foregoing excipients, carriers, orstabilizers will result in the formation of salts of the VEGF.

If the VEGF is to be used parenterally, therapeutic compositionscontaining the VEGF generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

Generally, where the disorder permits, one should formulate and dose theVEGF for site-specific delivery. This is convenient in the case ofwounds and ulcers.

Sustained release formulations may also be prepared, and include theformation of microcapsular particles and implantable articles. Forpreparing sustained-release VEGF compositions, the VEGF is preferablyincorporated into a biodegradable matrix or microcapsule. A suitablematerial for this purpose is a polylactide, although other polymers ofpoly-(a-hydroxycarboxylic acids), such as poly-D-(-)-3-hydroxybutyricacid (EP 133,988A), can be used. Other biodegradable polymers includepoly(lactones), poly(acetals), poly(orthoesters), orpoly(orthocarbonates). The initial consideration here must be that thecarrier itself, or its degradation products, is nontoxic in the targettissue and will not further aggravate the condition. This can bedetermined by routine screening in animal models of the target disorderor, if such models are unavailable, in normal animals. Numerousscientific publications document such animal models.

For examples of sustained release compositions, see U.S. Pat. No.3,773,919, EP 58,481A, U.S. Pat. No. 3,887,699, EP 1 58,277A, CanadianPatent No. 1176565, U. Sidman et al., Biopolymers 22, 547[1983], and R.Langer et al., Chem. Tech. 12, 98[1982].

When applied topically, the VEGF is suitably combined with otheringredients, such as carriers and/or adjuvants. There are no limitationson the nature of such other ingredients, except that they must bepharmaceutically acceptable and efficacious for their intendedadministration, and cannot degrade the activity of the activeingredients of the composition. Examples of suitable vehicles includeointments, creams, gels, or suspensions, with or without purifiedcollagen. The compositions also may be impregnated into transdermalpatches, plasters, and bandages, preferably in liquid or semi-liquidform.

For obtaining a gel formulation, the VEGF formulated in a liquidcomposition may be mixed with an effective amount of a water-solublepolysaccharide or synthetic polymer such as polyethylene glycol to forma gel of the proper viscosity to be applied topically. Thepolysaccharide that may be used includes, for example, cellulosederivatives such as etherified cellulose derivatives, including alkylcelluloses, hydroxyalkyl celluloses, and alkylhydroxyalkyl celluloses,for example, methylcellulose, hydroxyethyl cellulose, carboxymethylcellulose, hydroxypropyl methylcellulose, and hydroxypropyl cellulose;starch and fractionated starch; agar; alginic acid and alginates; gumarabic; pullullan; agarose; carrageenan; dextrans; dextrins; fructans;inulin; mannans; xylans; arabinans; chitosans; glycogens; glucans; andsynthetic biopolymers; as well as gums such as xanthan gum; guar gum;locust bean gum; gum arabic; tragacanth gum; and karaya gum; andderivatives and mixtures thereof. The preferred gelling agent herein isone that is inert to biological systems, nontoxic, simple to prepare,and not too runny or viscous, and will not destabilize the VEGF heldwithin it.

Preferably the polysaccharide is an etherified cellulose derivative,more preferably one that is well defined, purified, and listed in USP,e.g., methylcellulose and the hydroxyalkyl cellulose derivatives, suchas hydroxypropyl cellulose, hydroxyethyl cellulose, and hydroxypropylmethylcellulose. Most preferred herein is methylcellulose.

The polyethylene glycol useful for gelling is typically a mixture of lowand high molecular weight polyethylene glycols to obtain the properviscosity. For example, a mixture of a polyethylene glycol of molecularweight 400–600 with one of molecular weight 1500 would be effective forthis purpose when mixed in the proper ratio to obtain a paste.

The term “water soluble” as applied to the polysaccharides andpolyethylene glycols is meant to include colloidal solutions anddispersions. In general, the solubility of the cellulose derivatives isdetermined by the degree of substitution of ether groups, and thestabilizing derivatives useful herein should have a sufficient quantityof such ether groups per anhydroglucose unit in the cellulose chain torender the derivatives water soluble. A degree of ether substitution ofat least 0.35 ether groups per anhydroglucose unit is generallysufficient. Additionally, the cellulose derivatives may be in the formof alkali metal salts, for example, the Li, Na, K, or Cs salts.

If methylcellulose is employed in the gel, preferably it comprises about2–5%, more preferably about 3%, of the gel and the VEGF is present in anamount of about 300–1000 mg per ml of gel.

The dosage to be employed is dependent upon the factors described above.As a general proposition, the VEGF is formulated and delivered to thetarget site or tissue at a dosage capable of establishing in the tissuea VEGF level greater than about 0.1 ng/cc up to a maximum dose that isefficacious but not unduly toxic. This intra-tissue concentration shouldbe maintained if possible by continuous infusion, sustained release,topical application, or injection at empirically determined frequencies.

It is within the scope hereof to combine the VEGF therapy with othernovel or conventional therapies (e.g., growth factors such as aFGF,bFGF, PDGF, IGF, NGF, anabolic steroids, EGF or TGF-a) for enhancing theactivity of any of the growth factors, including VEGF, in promoting cellproliferation and repair. It is not necessary that such cotreatmentdrugs be included per se in the compositions of this invention, althoughthis will be convenient where such drugs are proteinaceous. Suchadmixtures are suitably administered in the same manner and for the samepurposes as the VEGF used alone. The useful molar ratio of VEGF to suchsecondary growth factors is typically 1:0.1–10, with about equimolaramounts being preferred.

5. Pharmaceutical Compositions

The compounds of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe VEGF variants hereof is combined in admixture with apharmaceutically acceptable carrier vehicle. Suitable carrier vehiclesand their formulation, inclusive of other human proteins, e.g., humanserum albumin, are described, for example, in Remington's PharmaceuticalSciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al. thedisclosure of which is hereby incorporated by reference. The VEGFvariants herein may be administered parenterally to subjects sufferingfrom cardiovascular diseases or conditions, or by other methods thatensure its delivery to the bloodstream in an effective form.

Compositions particularly well suited for the clinical administration ofVEGF variants hereof employed in the practice of the present inventioninclude, for example, sterile aqueous solutions, or sterile hydratablepowders such as lyophilized protein. It is generally desirable toinclude further in the formulation an appropriate amount of apharmaceutically acceptable salt, generally in an amount sufficient torender the formulation isotonic. A pH regulator such as arginine base,and phosphoric acid, are also typically included in sufficientquantities to maintain an appropriate pH, generally from 5.5 to 7.5.Moreover, for improvement of shelf-life or stability of aqueousformulations, it may also be desirable to include further agents such asglycerol. In this manner, variant t-PA formulations are renderedappropriate for parenteral administration, and, in particular,intravenous administration.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. For example, in the treatment of deep vein thrombosis orperipheral vascular disease, “bolus” doses, will typically be preferredwith subsequent administrations being given to maintain an approximatelyconstant blood level, preferably on the order of about 3 μg/ml.

However, for use in connection with emergency medical care facilitieswhere infusion capability is generally not available and due to thegenerally critical nature of the underlying disease (e.g., embolism,infarct), it will generally be desirable to provide somewhat largerinitial doses, such as an intravenous bolus.

For the various therapeutic indications referred to for the compoundshereof, the VEGF molecules will be formulated and dosed in a fashionconsistent with good medical practice taking into account the specificdisorder to be treated, the condition of the individual patient, thesite of delivery, the method of administration and other factors knownto practitioners in the respective art. Thus, for purposes herein, the“therapeutically effective amount” of the VEGF molecules hereof is anamount that is effective either to prevent, lessen the worsening of,alleviate, or cure the treated condition, in particular that amountwhich is sufficient to enhance the growth of vascular endothelium invivo. In general a dosage is employed capable of establishing in thetissue that is the target for the therapeutic indication being treated alevel of a VEGF mutant hereof greater than about 0.1 ng/cm³ up to amaximum dose that is efficacious but not unduly toxic. It iscontemplated that intra-tissue administration may be the choice forcertain of the therapeutic indications for the compounds hereof.

The following examples are intended merely to illustrate the best modenow known for practicing the invention but the invention is not to beconsidered as limited to the details of such examples.

EXAMPLES

Vascular Endothelial Growth Factor (VEGF) is a heparin binding growthfactor that may be clinically beneficial for revascularization ofischemic tissues. Significant differences in the pharmacological profileof Chinese hamster ovary (CHO) and E. coli-derived VEGF have beenobserved. The purpose of this study was to determine the molecular basisfor the pharmacologic differences between CHO and E. coli VEGF. Isolatedrat liver perfusion and whole animal pharmacokinetic studies were usedin conjunction with heparin column chromatography and mass spectralanalysis to characterize differences in clearance determinants. E. coliVEGF had a greater first pass hepatic extraction and higher volume ofdistribution when compared with CHO VEGF.

E. coli VEGF CHO VEGF CL (mL/min/kg) 11.5 4.0 Vdss (mL/kg) 1358.6 1263.9

Preincubation of E. coli VEGF with heparin reduced hepatic extractionsuggesting that heparin sulfate proteoglycan binding was in partresponsible for the rapid hepatic uptake. Using heparin columnchromatography, differences in the binding affinity between CHO and E.coli VEGF were noted. CHO VEGF appeared heterogeneous and elutedearlier. Mass spectral analysis of the reduced and carboxy methylatedCHO VEGF revealed internal proteolytic clips within the C-terminalheparin binding domain. E. coli VEGF behaved similarly to CHO VEGF invitro and in vivo following limited proteolysis. These resultsdemonstrate that the CHO VEGF was proteolytically clipped in theC-terminal heparin binding region which gave rise to reduced heparinbinding and hence, a slower clearance and lower volumn of distributionin vivo.

Comparison of the heparin binding properties of CHO and E. coli derivedVEGF: Three lots of CHO and two lots of E. coli-derived VEGF wereanalyzed using heparin column (Pharmacia) chromatography. Linear NaClgradients (150 mM to 2.0 M NaCl, 10 mM NaP pH 7.4, 0.01% T-20) were usedat a 0.5–1 ml/min flow rate for elution. The CHO lots appeared to beheterogeneous when compared to the VEGF produced in E. coli. CHO derivedVEGF appeared as a doublet with approximately 40% total peak areaeluting at 520 mM NaCl (peak 1) and 60% of the total peak area elutingat approximately 720 mM NaCl (peak 2). Additional peaks co-eluting withpeak 1 were apparent when shallower gradients were used. In contrast,the E. coli derived VEGF appeared as a single homogeneous peak followingheparin column chromatography under identical conditions. The E. coliVEGF had a slightly longer retention time, requiring approximately 745mM NaCl for elution.

Heparin binding differences are not related to sialic acid content:Studies were done to investigate the possibility that glycosylationheterogeneity gave rise to the multiple peaks observed in thechromatograms of CHO-VEGF following heparin chromatography. In the firststudy CHO VEGF was treated with neurominadase to remove sialic acid.Quantitative removal of sialic acid was verified by Dionex (L. Basa).The retention time of CHO VEGF on heparin was unchanged following sialicacid removal, suggesting that differences in peak 1 and peak 2 was notdue to differences in sialic acid content.

In the second study, peaks 1 and 2 were collected separately andcompared to unfractionated CHO by isoelectric focusing. Acidic bandswere represented in all peaks, however, there was a notable absence ofmore basic bands in the earlier eluting peak 1. These data providedfurther evidence that the multiple peaks observed following heparincolumn chromatography were not due to differences in sialic acidcontent. Instead, it appeared that peak 1 was lacking basic bands,rather than having additional acidic bands, when compared to peak 2. TheC-terminal region of VEGF is highly basic and known to be important forheparin binding, thus it was reasoned that the apparent heterogenity inthe heparin binding affinity of VEGF could be due to C-terminal removalof basic amino acids within the heparin binding domain.

C-terminal degradation of CHO VEGF: Peak 1 and peak 2 eluting fromheparin were collected separately following heparin columnchromatography and were analyzed by SDS-PAGE and LC-MS. For SDS-PAGEanalysis, the different fractions were radioiodinated to enhance thesensitivity of detection of various molecular forms of VEGF. Peak 1 andpeak 2 were run under reducing and non-reducing conditions. Both peaksappeared to be similar to a control under non-reducing conditions andall fractions ran at the expected molecular weight (43 kDA). However,heterogenity was apparent under reducing conditions. Multiple bandsranging in molecular weight from 15 to 20 kDA were apparent in the peak1 fraction, whereas peak 2 only had 1 minor band lower than the expectedmolecular weight of VEGF monomer.

Mass spec analysis of the N-glyconase, reduced and carboxymethylatedVEGF indicated that peak 2, the major peak represented VEGF 163/163.Peak 1 represented VEGF that had been further clipped within disulfiedbonds C-terminal to amino acids 110, 125, and 147. These data indicatethat the apparent heterogenity in the CHO VEGF are a result ofC-terminal processing with the heparin binding domain. Lastly, limiteddigestion of E. coli VEGF with carboxypeptidase B resulted in a peakthat co-eluted with CHO peak 2. Mass spec analysis of this peak revealedthat this peak was VEGF 163/163. A peak with similar retention time topeak 1 was observed following prolonged treatment (24 h) withcarboxypeptidase B. Thus E. coli VEGF can be made to “behave” like CHOVEGF with respect to heparin binding following limited C-terminaldigestion with CPB.

One milligram aliquots (200 μl ) of rhVEGF₁₆₅ lot D9837A were diluted to1 mL with 0.2 M NH₄HCO₃. Worthington PMSF treated CpB was added to therhVEGF,₁₆₅ solutions at an enzyme:substrate ratio of 1:50. The sampleswere digested at ambient temperature for 18 hours. The CpB digestedrhVEGF₁₆₅ samples were purified by heparin chromatography. Thepreparatively collected fractions were pooled, then concentrated, usinga Amicon Centricon-10 concentrator. The concentrated fractions werereformulated into 5 mM succinate, 275 mM trehalose by a Centricon-10concentrator. After the fractions were concentrated, 0.1% Tween-20 wasadded to the solutions. Protein concentration of each fraction wasdetermined by heparin chromatography and compared to peak areas of astandard solution of rhVEGF₁₆₅ D9837A.

Heparin Chromatographic Conditions:

-   Column: TosoHass Heparin-5PW column 7.5 mm×7.5 cm-   Flow rate: 1.0 mL/minute-   Detection: UV absorbance at 214 nm-   Mobile phase:    -   A: 10 mM NaH₂PO₄, 150 mM NaCl, pH 7.4    -   B: 10 mM NaH₂PO₄, 2 M NaCl, pH 7.4-   Gradient: 0% B to 80% B in 30 minutes linear    Large Scale Experiments:

Large scale experiments were performed on batched of 4 vial. Four vialsof M3RD587 were concentrated and reformulated in 0.2 m NH₄HCO₃ toapproximately 1.5 mL (7.7 mg/mL) using a Centricon-10 concentrator.Worthington PMSF treated CpB was added to the rhVEGF₁₆₅ solutions at anenzyme:substrate ratio of 1:50. The samples were digested at 37° C. for30 to 40 minutes. The CpB digested rhVEGF₁₆₅ samples were purified byheparin chromatography. The preparatively collected fractions werepooled, then concentrated using a Amicon Centriprep-10 concentrator. Theconcentrated fractions were reformulated into 5 mM succinate, 275 mMtrehalose using the same Centriprep-10 concentrator. After the fractionswere concentrated, 0.1% Tween-20 was added to the solutions. Proteinconcentration was determined by absorbance at 276 nm and amino acidanalysis.

Heparin Chromatographic conditions:

-   Column: Pharmacia 5 mL HiTrap Heparin column-   Flow rate: 2.0 mL/minute-   Detection: UV absorbance at 214 nm-   Mobile phase:    -   A: 10 mM NaH₂PO₄, 150 mM NaCl, pH 7.4    -   B: 10 mM NaH₂PO₄, 2 M NaCl, pH 7.4-   Gradient: 0% B to 80% B in 30 minutes linear    Effect of VEGF variants on ACE cell proliferation: Bovine adrenal    cortical endothelial (ACE) cells were plated on 6-well plates at    6000 cells/well and cultured in medium containing 10% fetal calf    serum. VEGF was added to the medium on the first day of culture at a    concentration of 1 nM. ACE cells were trypsinized on day 5 and    counted in a Casy-1 cell counter. Control cultures received diluting    buffer in place of VEGF. Data represent a mean of 5 replicate    samples (n=5). These data indicate that VEGF modified in the heparin    binding domain still retain biological activity (i.e., the ability    to stimulate endothelial cells to grow in culture).

Effect of heparin on VEGF binding to blood cells in rats and rabbits:The attached figure demonstrates that radioiodinated E. coli VEGF bindsto blood cells in rabbits and rats to a greater extent that CHO VEGF.Modifying the E. coli-derived VEGF by limited proteolysis (see EC1+2)decrease this interaction. The fact that the 110/110 form of VEGF doesnot associate with the blood cell pellet provides further evidence thatVEGF binding to RBCs appears is mediated by the heparin binding domain.These data further demonstrate that modification of the heparin bindingdomain could limit the loss of rhVEGF to nonspecific binding on varioustissues (in this case the example being blood cells).

Concluding Remarks

The foregoing description details specific methods which can be employedto practice the present invention. Having detailed such specificmethods, those skilled in the art will well enough know how to devisealternative reliable methods at arriving at the same information inusing the fruits of the present invention. Thus, however detailed theforegoing may appear in text, it should not be construed as limiting theoverall scope thereof; rather, the ambit of the present invention is tobe determined only by the lawful construction of the appended claims.All documents cited herein are hereby expressly incorporated byreference.

1. A pharmaceutical composition comprising a vascular endothelial cellgrowth factor (VEGF) variant polypeptide and a pharmaceuticallyacceptable carrier, wherein said variant polypeptide differs from nativeVEGF comprising a C-terminal heparin binding domain by comprising astructural alteration of the C-terminal heparin binding domain resultingin modified pharmacokinetic properties of said variant as compared tonative VEGF, wherein said variant binds heparin at a lower affinity thansaid native VEGF, and wherein said structural alteration is a truncationof said heparin binding domain.
 2. A pharmaceutical compositioncomprising a vascular endothelial cell growth factor (VEGF) variantpolypeptide and a pharmaceutically acceptable carrier wherein saidvariant polypeptide differs from native VEGF by containing a C-terminalheparin binding domain structural alteration such that said domain bindsheparin at a lower affinity than native VEGF comprising a heparinbinding domain, resulting in said VEGF variant polypeptide having areduced clearance rate compared with native VEGF, and wherein saidstructural alteration is a truncation of said heparin binding domain. 3.The composition according to claim 1 or 2, wherein said truncationcomprises a reduction in positively charged amino acids.
 4. Thecomposition according to claim 3, wherein said C-terminal heparinbinding domain of said VEGF variant polypeptide has an alteredconformational structure as compared to said native VEGF.
 5. Thecomposition according to claim 1 or 2, wherein said C-terminal heparinbinding domain is truncated at about amino acid
 120. 6. The compositionaccording to claim 1 or 2, wherein said C-terminal heparin bindingdomain is truncated at about amino acid
 125. 7. The compositionaccording to claim 1 or 2, wherein said C-terminal heparin bindingdomain is truncated at about amino acid 147.